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Revista de diagnóstico y biomarcadores moleculares

Volumen 9, Asunto 2 (2018)

Artículo de investigación

Assessment of the Correlation between Serum Prolidase and Alpha-Fetoprotein Levels in Egyptian Patients with Hepatocellular Carcinoma

Mohamed Osama Aly, Osama Aboelfotoh Aly, Hossam Abdul-Aziz Mahmoud, Nourhan Assem Ali

Background: Hepatocellular carcinoma (HCC) accounts as the sixth most common neoplasm on a global scale and the third most lethal with >600,000 deaths per year worldwide. Despite regular surveillance to detect small HCC, HCC is often diagnosed at advanced stage, after the symptoms related to HCC have appeared, and the 5-year survival rate for patients is only 7%. If HCC could be diagnosed at an early stage, potentially curative option, such as resection, ablation, and transplantation may be considered. Prolidase is an important enzyme that cleaves the bonds of dipeptides containing proline (X-pro), and plays a vital role in collagen turnover, matrix remodeling and cell growth. Metastatic tumor cell produce enhanced number of proteases that enable them to penetrate basement membrane and extracellular matrix. Therefore, tumor progression might depend on the breakdown of collagen and other extracellular matrix proteins.
Aim of the work: Assess the possible diagnostic role of serum prolidase compared to alpha- fetoprotein which is the slandered marker used for diagnosis of HCC.
Methods: The study was conducted upon 90 subjects who were divided into three groups: group I included 40 patients with liver cirrhosis and hepatocellular carcinoma, group II included 40 patients with HCV related liver cirrhosis without HCC, group III had 10 healthy subjects as controls. Plasma prolidase was measured by Enzyme Linked Immunosorbent assay (ELISA) using recombinant human peptidase D/prolidase (PEPD) ELISA kit lot. Results: In this study, the serum levels of serum prolidase were highest in patients of group I with HCC compared to those with liver cirrhosis and the control groups. Also, prolidase values increased with tumor number, overall size but not with vascular invasion nor with BCLC.
Conclusion: There was a direct significant correlation between serum prolidase level and serum AFP level in HCC patients. Serum prolidase levels directly correlated to the tumor number and overall size so it has a good prognostic value.

Artículo de investigación

Latent Transforming Growth Factor-beta Binding Protein-1 as a Diagnostic Biomarker for the Detection of Hepatocellular Carcinoma

Bishoy El-Aarag, Hend Saad, Olfat Hendy, Mohamed Abdel-Samiee and Magdy Zahran

Background: Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancerrelated death worldwide. The current study aims to evaluate the diagnostic role of latent transforming growth factorbeta binding protein-1 (LTBP-1) as a biomarker to distinguish hepatocellular carcinoma (HCC) from patients with liver cirrhosis. Methods: The study carried out on 90 individuals classified to healthy individuals (n=20), liver cirrhosis (n=30), and HCC (n=40). The serum level of LTBP-1 was measured by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristics (ROC) curves and area under the curve (AUC) were calculated. Results: The level of LTBP-1 was significantly higher in HCC patients than healthy and patients with cirrhosis. Furthermore, there was a significant (p<0.001) association between the level of LTBP-1 and CLIP and BCLC in HCC patients. Moreover, LTBP-1 levels were significantly (p=0.01) associated to child pugh grade in patients with cirrhosis and HCC. ROC curve analyses revealed that LTBP-1 showed a better diagnostic performance (AUC=0.970, Sensitivity: 82.50%, Specificity: 96.67%, PPV: 97.06%, NPV: 80.56%) in distinguishing HCC from cirrhosis patients, compared to AFP (AUC=0.810, Sensitivity: 62.50%, Specificity: 93.33%, PPV: 92.59%, NPV: 65.12%). Conclusion: These findings suggested that LTBP-1 may be a promising biomarker for distinguish HCC from liver cirrhosis patients.

Artículo de investigación

Detection of Oxidative Stress in Buccal Cells using iSWAB Tubes

Marcel Bassil, Aya Al Atat, and Mariam Ibragim

Oxidative stress is the result of an imbalance in pro-oxidant/antioxidant homeostasis in favor of oxidants leading to the oxidation of DNA, lipids, and proteins. In this experiment, we investigated the oxidative profile of iSWAB-Mawi protein tubes by examining their ability to detect the protein oxidation biomarkers: nitrotyrosine and S-nitrosocysteine in buccal cells in a sample of 40 participants, equally divided between Lebanese University and Benta Pharma Industries. Protein concentrations were determined by Bradford assay in order to then be analyzed for the presence of nitrotyrosine and S-nitrosocysteine by western blot. Results showed the presence of S-nitrosocysteine and nitrotyrosine in buccal cells reflecting the ability of iSWAB-Mawi protein tubes to detect those protein oxidation biomarkers.

Artículo de investigación

Comparison of Immunohistochemistry Assay Results with Gene Expression Profiling Methods for Diffuse Large B-Cell Lymphoma Subtype Identification in Matched Patient Samples

Michael Schaffer, Shalini Chaturvedi, JD Alvarez, Sandy Frans, Regina Aquino, Brett Hall, Mark Wildgust and Sriram Balasubramanian

Background: The cell of origin (COO) in diffuse large B-cell lymphoma (DLBCL) has prognostic importance. While the COO was originally classified into germinal center B-cell (GCB) or activated B-cell (ABC) subtypes by microarray analysis, routine use was not practical. Immunohistochemistry (IHC)–based methods are widely used with varying results due to lack of standardization. Several classification methods have been developed recently; understanding the concordance between these and existing methods is essential to their practical application. Therefore, we evaluated concordance between 3 commercial assays: a standardized Hans-based IHC method and 2 gene expression profiling (GEP) methods and compared these to the accepted microarray classification.
Methods: 137 DLBCL-confirmed tumor samples were evaluated using a standardized Hans-based IHC method for GCB or non-GCB subtype, by a published microarray-based assay, a digital gene expression-based Lymphoma Subtyping Test (LST) and a next-generation sequencing-based assay (EdgeSeq COO). Subtype calls from the 3 GEP methods were harmonized to “GCB” or “non-GCB” and assessed for concordance.
Results: Concordance between the Hans-based IHC assay and the microarray-based assay, LST assay and EdgeSeq assay was 79.6% (N=137), 80.0% (N=125) and 78.2% (N=64), respectively; the positive percent agreement (PPA) in non-GCB was 88.9%, 87.0% and 78.1%, respectively. Concordance for the Hans-based IHC assay versus GEP methods was especially high for direct GCB calls (91.0%, 88.3% and 78.1% for microarray, LST and EdgeSeq COO methods, respectively). The newly developed GEP assays performed well against the microarray GEP method, against which they were calibrated (concordance 93.7% and 87.5% and PPA 94.3% and 92.9%, respectively, for LST and EdgeSeq COO).
Conclusion: These results demonstrated good consistency between various platforms for stratification of DLBCL into COO subtype classifications. Application of a standardized Hans-based IHC assay offers a robust, rapid and easily accessible platform to classify DLBCL into prognostically important subtypes.

Artículo de investigación

Evaluation of Inhibitory Zone Diameter (IZD), Phytochemical Screening, Elemental Composition and Proximate Analysis of Crude Cleistopholis Patens (Benth) on Infectious Clinical Isolates

Oludare Temitope Osuntokun

This purpose of this study is to determine the Inhibitory zone diameter (Izd), phytochemical screening, elemental composition and proximate analysis of crude Cleistopholis patens leaf, bark, aqueous and ethanol extracts on selected pathogenic isolates. The leaf and bark of Cleistopholis patens plants were obtained from a location in the southwestern part of Nigeria, in the tropical rainforest of Ikare Akoko, Ondo state and Ile Ife, Osun state, Nigeria. The six (6) bacterial isolates include Escherichia coli, Salmonella typhi, Candida albicans, Klebsiella pneumonia, Aspergilus flavus, and Staphylococcus aureus. Bacteria isolate were identified by various biochemical tests and morphological characteristics. Four concentrations were used to determine the Inhibitory zone Diameter namely; 60, 30, 15, and 7.5 mg/ml and two controls were used namely; Ciprofloxacin and Metronidazole. All clinical organisms show higher zones of inhibition at 60 mg/ml and lower zones of inhibition at 7.5 mg/ml while control shows 28.0 at 30 mg/ml zones of inhibition on Klebsiella pneumoniae, while Salmonella typhi and Aspergilius flavus shows lower zone of inhibition at 30 mg/ml. In qualitative phytochemical screening of Cleistopholis patens stem bark extract, it was observed that alkaloid and anthraquinone are negative while cardiac glycoside, steroid, phenol, tannins, saponin, and flavonoids were positive. in qualitative phytochemical screening of Cleistopholis patens Leaf extract, alkaloid, steroid, and anthraquinone are negative while cardiac glycoside, phenol, tannins, saponin and flavonoids are positive. In the elemental determination of Cleistopholis patens stem bark extract Zinc (Zn) has 28.0 mg/g which is the highest value while copper has the lowest value which is 0.03 mg/g. In Cleistopholis patens leaf extract, Calcium (Ca) has 25.32 mg/g which is the highest while copper has the lowest which is 0.03 mg/g. while Lead (Pb) was not detected. In Cleistopholis patens stem bark extract (Phylate) has the highest anti-nutrients which is 17.30% while Alkaloids has the lowest anti-nutrients which are 1.23%. In Cleistopholis patens leaf extract Phylate, has the highest anti-nutrients, value 17.27% while Alkaloids has the lowest anti-nutrients value, 1.25%. In Cleistopholis patens stem bark extract, carbohydrate has the highest percentage which is 46.69% and fat has the lowest percentage which is 6.48%. In Cleistopholis patens leaf extract, carbohydrate has the highest percentage which is 48.91% and fat has the lowest percentage which is 8.53%. Cleistopholis patens is useful for the treatment of infectious diseases.

Comunicación corta

Sheehan’s Syndrome: A Rare but Potentially Life-Threatening Complication of Postpartum Haemorrhage

Monika Thakur, Kunal Mahajan, Sandeep Sharma and Gunjan Gupta

Sheehan’s syndrome (SS), also known as postpartum pituitary gland necrosis, is a rare but potentially life-threatening complication of postpartum haemorrhage. The diagnosis of Sheehan’s syndrome is based on the features of hormone deficiency, a suggestive obstetric history and decreased levels of basal hormones. Diagnosis of pan-hypopituitarism is straightforward, but partial deficiencies are often difficult to elicit. SS is a frequent cause of hypopituitarism in underdeveloped countries. The clinical features of hypopituitarism are often subtle, and years may pass before the diagnosis is made following the inciting delivery. History of postpartum haemorrhage, failure to lactate and cessation of menses are important clues to the diagnosis.

Artículo de investigación

Urinary Neutrophil Gelatinase- Associated Lipocalin in Children with Idiopathic Nephrotic Syndrome: A Biomarker to Differentiate Between Steroid Sensitive and Resistant Forms of Disease

Om P Mishra, Santosh K Mehta, Pradeep Srivastava, Abhishek Abhinay, Rajniti Prasad and Vineeta V Batra

Background: Objectives of the study were to find out the ability of urinary neutrophil gelatinase- associated lipocalin (NGAL) as a biomarker to differentiate between steroid resistant nephrotic syndrome (SRNS) and steroid sensitive nephrotic syndrome (SSNS), and also to observe variation in levels, if any, between focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) in SRNS patients.
Methods: The study included 63 patients of idiopathic nephrotic syndrome (19 SSNS in relapse, 19 SSNS in remission and 25 SRNS), aged 2.5-16 years, along with 17 controls. Urinary NGAL was measured by ELISA and the values were normalised with urinary creatinine.
Results: Median urinary NGAL/creatinine level was significantly raised in SRNS as compared to SSNS relapse, SSNS remission and controls (p< 0.001). A cut-off level of NGAL/creatinine >18.3 ng/mg had sensitivity of 84%, specificity of 97.4% and area under the curve of 0.935 (p< 0.001, 95% confidence interval 0.871-0.998) to differentiate SRNS from SSNS patients. Median urinary NGAL/creatinine value was significantly raised in children with FSGS as compared to MCD (p=0.003) in SRNS patients. It had significant positive correlations with duration of illness (r=0.342, p=0.006), urine protein creatinine ratio (r=0.594, p< 0.001) and negative correlation with serum albumin (r=0.470, p<0.001).
Conclusion: Urinary NGAL/creatinine level correlated with activity of the disease and it can distinguish not only SRNS from SSNS but also FSGS and MCD histopathological sub-types of SRNS in children.

Reporte de un caso

Molecular Diagnostic Approach Prevails Superior Over Conventional GelElectrophoresis Method in Detecting Sickle Cell Anemia

Khadiza Begum, Mohammad Abdul Mannan, Mousumi Sanyal, Md. Ismail Hosen, Sajib Chakraborty and Hossain Uddin Shekhar

Sickle cell anemia is defined as homozygosity caused by the mutation of the glutamic acid residue to valine in the β-globin gene. Sickle cell disease is an increasing global health burden with the estimated number of patients increasing in a concerning manner. Here we report a very interesting and clinically insightful case of hemoglobinopathy which was initially suspected to be Hb S/D Punjab- a rare type of hemoglobinopathy as diagnosed by the hemoglobinelectrophoresis technique. Despite the diagnosis report indicating the rare Hb S/D Punjab, the uncharacteristic clinical presentation of the patient which was not coherent with the classical symptoms of Hb S/D Punjab, forced the clinicians to turn their attention to molecular diagnosis. To clarify the etiology of the clinical case, a sequencing-based molecular diagnosis approach was adopted that revealed the mutational signature of sickle cell anemia (SCA). This case can be regarded as a prominent example where the molecular techniques lead to the correct diagnosis matching with the clinical symptoms while the conventional diagnostic approach failed.

Comunicación corta

Metabolites-Mitochondria-Microglia (Mmma): Therapeutic Stategy for Treatment of Neural Inflammation and Neurodegeneration

Corrado Angelini and Arianna Di Stadio

Patients with mitochondrial disease often present with prominent neurologic symptoms; the alteration of mitochondrial metabolism produces an excess of ROS and cytokines that appeared implicated in the activation of microglia; similar considerations apply to Amyotrophic Latera Sclerosis (ALS) and other neurodegenerative disorders. The microglia polarization by mitochondrial metabolism modulation could be considered an option to treat the neuroinflammation and neurodegenerative disorders, similar to macrophage action in case of muscle inflammation and atrophy. Furthermore, thanks to the availability of several biomarkers including mitochondrial microRNAs it should be possible to monitor metabolic state and microglia mitochondrial metabolic shift.

Artículo de investigación

Evaluation of Aberrant Methylation of RASSF1A , BCL-XL , ITGA6 , TCF3 and SNAIL2 Genes in Peripheral Blood Leukocyte DNA in Breast Cancer Patients

Zahra Niki Boroujeni, Atefeh Shirkavand, and Ahmad Aleyasin

Purpose: Nowadays, breast cancer is the most common cancer in women that caused by defects in the signaling mechanisms that control cell proliferation and apoptosis. Recent findings suggest that epigenetic alterations are the key factors in the development of breast cancer. Methylation changes occur within CpG islands of promoters and induce gene silencing. Abnormal methylation can be used as a potential biomarker for diagnosis of various diseases including cancer. In this study, methylation changes of RASSF1A, TCF3, BCL-XL, SNAIL2 and ITGA6 genes were assessment as epigenetic biomarkers of breast cancer.
Methods: 70 breast cancer samples and 70 normal samples were selected and identified with different Clinical and pathological data, which might be related with methylation changes. Breast cancer patients and normal blood samples were collected, and DNA was extracted from white blood cells. DNA samples were digested using methylationsensitive restriction enzymes to identify methylated sites. Unlike hypomethylated positions, hypermethylated sites were not digested using these enzymes, thus replication occurs by PCR reaction.
Results: RASSF1A and TCF3 (in some cases) were significantly hypermethylated in breast cancer cases (P<0.05) compared to normal samples. ITGA6 was significantly hypomethylated in breast cancer cases (P<0.05) compared to normal samples. According to statistical analysis, no significant correlation was observed between methylation changes and clinical factors (stage of disease, age of patients, Estrogen Receptor (ER), Progesterone Receptor (PR), and human epidermal growth factor 2 (HER2) status) in patients with breast cancer (P>0.05) except RASSF1A gene ethylation changes that shown reverse correlation with age of patients (P<0.05).
Conclusion: This study demonstrated that RASSF1A, ITGA6 and TCF3 genes methylation status were changed during breast cancer and they can be used as molecular biomarkers for breast cancer diagnosis.

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